Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins†,Analyst

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Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins†
Analyst ( IF 3.6 ) Pub Date : 2017-12-01 00:00:00 , DOI: 10.1039/c7an01508a
Lenka Hromadkova _{1,

2,

3,

4,

5} , Rudolf Kupcik _{1,

2,

3,

4,

5} , Marie Vajrychova _{5,

6,

7,

8,

9} , Petr Prikryl _{5,

10,

11,

12,

13} , Andrea Charvatova _{1,

2,

3,

4,

5} , Barbora Jankovicova _{1,

2,

3,

4,

5} , Daniela Ripova _{5,

14,

15} , Zuzana Bilkova _{1,

2,

3,

4,

5} , Marcela Slovakova _{1,

2,

3,

4,

5}

Affiliation

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni²⁺, or Co³⁺ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

中文翻译：

装有激酶的磁珠可用于肽和蛋白质的连续体外磷酸化†

翻译后修饰，包括磷酸化，极大地影响了蛋白质的生理功能，尤其是那些天然存在并与许多神经退行性疾病有关的蛋白质。但是，此类蛋白质的结构和功能研究需要完全定义的磷酸化，包括非生理性的磷酸化。因此，激酶ERK2和GSK-3β被固定在具有羧基，醛基，Ni ²⁺或Co ³⁺官能团的各种超顺磁珠上，以期在体外有效地磷酸化肽和蛋白质。。特定合成肽的完全磷酸化证实珠子已成功加载激酶。值得注意的是，共价固定在羧化SeraMag磁珠上的酶在重复使用后仍保持活性，在10次使用后，GSK-3β的残留活性为99.5±0.34％，ERK2为36.2±2.01％。珠还用于顺序磷酸化重组tau，重组tau在体内是阿尔茨海默氏病的生物标记。因此，首次展示了由两个固定在磁珠上的完全活性激酶组成的系统。与可溶性酶相比，磁珠更易于处理，可重复使用，因此成本较低。重要的是，这些珠也方便从反应中除去，以使磷酸化产物的污染最小化或与其他激酶交换。

更新日期：2017-12-01

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